This proposal seeks to understand the structure and mechanism of action of cathelicidin, a mammalian antimicrobial peptide. Our laboratory has shown that expression of the murine cathelicidin (CRAMP) is critical to resistance against invasive skin infection caused by Group A Streptococcus (GAS). In this way, cathelicidins are the only class of antimicrobial peptide proven to be a vital part of mammalian innate defense against disease. Prior work also suggests that cathelicidins act by directly killing bacteria and by activating host cell responses involved in wound repair and leukocyte recruitment. Thus, the cathelicidins represent a vital part of defense of the skin against microbial infection and are likely to participate in a coordinated response with elements of the cutaneous adaptive immune system. The mechanism of action of these molecules is generally unknown. Evolutionarily conserved domains in the immature pro-form of the antimicrobial peptide have not been thoroughly investigated but likely have functions related to the cystatin superfamily to which these domains are related. Due to the recently recognized importance of cathelicidins but relative lack of information regarding their structure, function and regulation, the specific aims of this work are to: 1) Investigate the function of distinct structural domains of cathelicidins by recombinant production of the intact pre-processed form of this protein, determine crystal structure, and proceed in a structure/function analysis of active domains. 2) Determine how cathelicidins function as antimicrobial peptides by a bacterial molecular genetic approach. 3) Determine the mechanisms of host response to microbial pathogenesis by identification of molecules released by bacteria that trigger keratinocyte cathelicidin production. Successful completion of these aims will advance our understanding of a critical part of the innate immune system and establish information that will aid design of new diagnostic and therapeutic approaches to skin defense against infection.